The dengue viral genome, an 11 kb strand of positive-sense RNA, contains genes for the three structural proteins, capsid (C), pre-membrane(pre-M) and envelope (E), followed by a series of 7 non-structural protein genes. Earlier, we constructed a full-length cDNA copy of the entire dengue 4 (D4) genome, which yields infectious full-length RNA transcripts. Subsequently the C-preM-E genes of the full-length clone were replaced with the C-preM-E genes of the Western Pacific strain of D1 virus or the C-preM-E or preM-E genes of the parental New Guinea C strain of D2 virus, and used as templates to create chimeric D1/4 and D2/4 viruses. Cells infected with these chimeras produced the appropriate authentic D1 or D2 structural proteins. Two chimeric viruses were tested in rhesus monkeys to determine their potential usefullness as vaccines. Initially, monkeys were immunized with either D4 virus, wild-type D1 or D2, or chimeric D1/4 or D2/4 virus. Monkeys inoculated with D4 developed minimal viremia and were not protected against D1 or D2 challenge. Those given D1 developed 2-3 days of viremia, had a high-titer neutralizing antibody response, and were protected against subsequent D1 challenge. D1/4 recipients had little (1 day) or no viremia; three had a strong D1 neutralizing antibody response and developed little (1 day) or no viremia following subsequent D1 challenge, while the fourth monkey had a weak antibody response to immunization and had 4 days of viremia post-challenge. Each of the monkeys infected with D2 or D2/D4 developed viremia (4-6 days vs. 1-5 days, respectively). Each monkey developed a high-titer D2 antibody response and were completely protected against D2 challenge. In a second experiment, monkeys were immunized with D4 or with a mixture of the chimeric D1/D4 and D2/D4 viruses, as a prototype bivalent vaccine. The D4 recipients became viremic when challenged subsequently with D1 or D2, but each of the monkeys immunized with D1/4 plus D2/4 developed high- titer neutralizing antibodies against both D1 and D2 and were protected against challenge with D1 or D2. Our results suggest that a mixture of suitably engineered chimeric viruses might serve together as a safe and effective live dengue vaccine.